2f peracetyl fucose

To inhibit the synthesis of fucosylated structures, a competitive inhibitor of fucosyltransferases, the 2F-Peracetyl-Fucose (Merck) was used. This inhibitor enters the cell by passive diffusion and is metabolized into a GDP-fucose analog (2F-fucose) to form an inhibitory substrate of fucosyltransferases^{60 (link)}. Cells were seeded in 96-well plates or 25 cm² flasks and treated with 500 µM 2F-Peracetyl-Fucose (Merck) solubilized in DMSO in the adequate medium for 3 days, replacing the medium with fresh drug daily. Negative controls were performed by adding the DMSO vehicle only to the culture medium. Afterwards, treated cells were phenotyped by flow cytometry or infected by RVs.

KIAA1324 and its mutants were cloned into the pCMV-3HA vector (Clontech, California, USA) as previously described [13 (link)]. Asp (N) to Gln (Q) amino acid exchange mutation in putative N-glycosylation sites was performed by a DpnI-site directed mutagenesis method using mutagenic primers [21 (link)]. Anti-HA (Santa Cruz Biotechnology, Texas, USA, sc-7392) and anti-PARP (Santa Cruz Biotechnology, sc-8007), anti-caspase-3 (Cell Signaling Technology, Massachusetts, USA, #9662), anti-caspase-7 (Cell Signaling Technology, #9494), anti-phosphor-JNK (Cell Signaling Technology, #4668), anti-JNK (Cell Signaling Technology, #9252), anti-GRP78 (Abcam, Cambridge, United Kingdom, ab21685), anti-PDI, anti-β-actin (Abcam, ab8227), anti-KIAA1324 (Sigma, Missouri, USA, SAB2900912) and α-tubulin (Sigma, T5168) antibodies were used. 2F-Peracetyl-Fucose (Sigma, #344827), SP600125 (Sigma, S5567), SB203580 (Sigma, S8307), U0126 (Sigma, U120), tunicamycin (Sigma, T7765), and doxycycline (Sigma, #24390-14-5) chemicals were used.

Human HEK293T, MDA-MB-231, and mouse mammary carcinoma 4T1 cells, were bought from the American Type Culture Collection (Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibico) at 37 °C under 5% CO₂. HCC1806 cells were maintained in RPMI-1640 medium supplemented with 10 % FBS at 37 °C under 5% CO₂. All the cells were authenticated using short-tandem repeat profiling, and tested negative for mycoplasma contamination. Compounds MG132 (S2619, Selleck Chemical), cycloheximide (BS168A, Biosharp), Thiamet G (HY-12588, MCE), PUGNAc (A7229, Sigma), tunicamycin (T7765, Sigma), 2F-Peracetyl-Fucose (344827, Sigma), swainsonine (16860, Cayman Chemical), and deoxymannojirimycin (D9169, Sigma) were obtained commercially.